Duchenne muscular dystrophy is the most common monogenic pediatric inborn error, affecting one in 3,500 live born males world-wide. The most promising molecular therapeutic approach for DIVID is systemic delivery of anti-sense oligonucleotides (AOs), where the drugs alter mRNA splicing, and converting out-of-frame loss-off function transcripts to in-frame transcripts capable of producing semi-functional (Becker-like) dystrophin. In the large animai (dog) model of Duchenne dystrophy, we have shown that intravenous (iV) delivery of high dose morpholino AOs is able to rescue dystrophin protein production to about 20% of wild-type levels, and cause stabilization or improvement of multiple functional, histological, and imaging outcome measures (Yokota et al. 2009). Initial human clinical trials using morpholino AO directed at exon 51 have shown that both direct intramuscular injection and IV administration can result in de novo dystrophin production. However, it is anticipated that repeated doses of 20 mg/kg - 100 mg/kg are likely required for sustained efficacy. GLP toxicity studies in mice and non-human primates, sponsored by the applicants, have shown that high dose weekly IV injections can lead to accumulation of drug in kidney proximal tubule cells. This accumulation resolved after termination of dosing, and there were no elevation of standard markers of kidney damage. However, dosing of DIVID patients will need to be life-long, and there has been no optimization of dosing schedules or drug concentrations that balance efficacy in muscle vs. kidney accumulation. Here, we bring together an interdisciplinary team to define the therapeutic window of morpholino dosing. Project 1 collaborates with an ongoing dose-ranging clinical trial to monitor kidney toxicity through urine biomarkers and shed renal cells. Project 2 defines the effects of AO concentrations, and dosing regimen on drug accumulation in kidney tubule cells, and also carries out a biomarker discovery program to define sensitive and reliable urine biomarkers for morpholino-associated cell damage. Project 3 defines the optimal dosing regimen able to provide sustained clinical efficacy in the mouse model of muscular dystrophy, using an established murine drug-testing core. A kidney toxicology assessment core supports these projects. PUBLIC HEALTH RELEVANCE: This project will provide clinical samples to monitor kidney accumulation of morpholino AO with chronic high dose IV treatment in boys with DMD. This project will integrate the pre-clinical and clinical data from the entire U54 program to derive inform optimized dosing and therapeutic index. The treatment of DMD using high dose IV morpholino is the most promising strategy for therapeutics of DMD. The possible consequences of long-term chronic treatment are not known. The data from this Project 2 will help define intervention targets and test tools for effective treatment of DMD using antisense morpholino. We will determine an optimized therapeutic window using the mouse efficacy and rat toxicity data above, including renal function tests, renal histology and ultrastructure, urine epithelial cel assays, and urine proteomic biomarker assays through Project 2 and Core B. Use of PMOs to correct genetic defects is rapidly advancing to several other human diseases such as FSHD, Cystic fibrosis, myotonic dystrophy, LGMD etc. Therefore, the data generated from this proposal have much broader implications than Duchenne muscular dystrophy.